Neural Stem Cells Direct Axon Guidance via their Radial Fiber Scaffold

To determine the genes regulated by Fezf2 during neocortical development, we performed RNA-seq on Fezf2 +/+; Emx1-cre (Fezf2 wildtype) and Fezf2 fl/+; Emx1-Cre (Fezf2 Knockout) neocortices isolated from the P0 mice. After RNA isolation from the dissected tissue the samples were processed for library preperation using Illumina Poly-A RNA kit. Fezf2 can reprogram the upper layer neocortical neurons to form corticospinal tract. To understand the key transcriptomic changes driven by Fezf2 that drive this reprogramming, we performed in RNA-seq on the cells over-expressing Fezf2 and Gfp or Gfp. At E15.5, in utero electroporations targeting cortical plate, were performed in pregnant mice with Fezf2 and Gfp plasmids or with Gfp plasmid alone, which was used as control. At P3, the Gfp positive cells were FAC sorted from Fezf2 and Gfp, or Gfp cortices and processed for library preparations using NUGEN Ovation® V2 system. Libraries were sequenced using Illumina 2000 platform to generate 75bp single reads. At least 10 million uniquely mapped reads were obtained for each sample. To determine the genes directly regulated by FEZF2, we performed the ChIP-Seq on N2A cells that were transfected to over express FEZF2 using pCAG- Fezf2-V5 and pCAG-V5. After 48 hr, of transfection, cells were fixed and processed for ChIP seq. Libraries were prepared using Illumina TruSeq ChIP Libary Preparation Kit. Determining the unique genes regulated by Fezf2 during the neocortex development by RNA-seq and ChIP-seq ChIP antibody information: V5 Tag Monoclonal Antibody (ThermoFisher Scientific, Cat# R960-25, RRID:AB_2556564)为鉴定新皮层发育过程中受Fezf2调控的基因，我们对取自P0小鼠的Fezf2 +/+; Emx1-cre（Fezf2野生型）与Fezf2 fl/+; Emx1-Cre（Fezf2敲除型）新皮层组织开展了RNA测序（RNA-seq）。从解剖分离的组织中提取RNA后，使用Illumina Poly-A RNA建库试剂盒对样本进行文库制备。Fezf2可将上层新皮层神经元重编程为皮质脊髓投射神经元。为解析Fezf2介导该重编程过程的关键转录组变化，我们对过表达Fezf2与绿色荧光蛋白（Gfp）或仅过表达Gfp的细胞进行了RNA测序。在胚胎期15.5天（E15.5），我们对妊娠小鼠实施靶向皮层板的子宫内电转，分别导入Fezf2与Gfp质粒，或仅导入Gfp质粒作为对照。出生后第3天（P3），通过荧光激活细胞分选（FAC）从共转Fezf2与Gfp或仅转Gfp的皮层组织中分离Gfp阳性细胞，随后使用NUGEN Ovation® V2建库系统完成文库制备。采用Illumina 2000测序平台对文库进行测序，生成75bp单端读长序列，每个样本至少获得1000万条唯一比对读段。为鉴定Fezf2直接调控的靶基因，我们利用转染了pCAG-Fezf2-V5与空载体pCAG-V5的N2A细胞开展了染色质免疫沉淀测序（ChIP-seq）。转染48小时后固定细胞，完成ChIP测序文库制备，所用试剂盒为Illumina TruSeq染色质免疫沉淀建库试剂盒。ChIP所用抗体信息：V5标签单克隆抗体（赛默飞世尔科技，货号：R960-25，RRID:AB_2556564）